According to WHO statistics, as many as 170 million people worldwide are infected by hepatitis C virus (HCV), a viral infection of the liver. 75 to 85% of persons infected with HCV progress to chronic infection, approximately 20% of these cases develop complications of chronic hepatitis C, including cirrhosis of the liver or hepatocellular carcinoma after 20 years of infection. The current recommended treatment for HCV infections is a combination of interferon and ribavirin drugs, however the treatment is not effective in all cases and liver transplantation is indicated in hepatitis C-related end-stage liver disease. At present, there is no vaccine available to prevent HCV infection, therefore all precautions to avoid infection must be taken.
Thus, patient care, as well as the prevention of transmission of Hepatitis C Virus (HCV) by blood and blood products or by close personal contact requires extreme vigilance using sensitive detection assays. This creates a need for specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products. Serological determination of HCV exposure relies on the detection of anti-HCV antibodies present in human blood plasma or sera. These anti-HCV antibodies are directed against a number of distinct structural and non-structural proteins encoded by the virus.
The HCV virus is a (+) sense single-stranded enveloped RNA virus in the Hepacivirus genus of the Flaviviridae family. The viral genome is approximately 10 kb in length and encodes a 3011 amino acid polyprotein precursor. The HCV genome has a large single open reading frame (ORF) coding for a unique polyprotein. This polyprotein is co- and post-translationally processed by cellular and viral proteases into three structural proteins, i.e., core, E1 and E2 and at least six non-structural NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins. (Choo et al., Science 244: 359-362 (1989)).
There are commercially available assays that determine whether a subject has been exposed to HCV. These serological assays typically use an indirect format in which anti-HCV antibodies are captured by recombinant HCV antigens present on a solid phase, followed by detection of the anti-HCV antibody by a labeled anti-human antibody conjugate. While some of the antigenic regions of HCV have been identified, peptides and recombinant proteins from these regions exhibit a variable degree of sensitivity and selectivity in detection and diagnosis of HCV carriers.
For example, HC43 is one such recombinant protein used for the detection of HCV antibodies in human serum or plasma. HC43 contains the C33 region of the NS3 protein (HCV-1 amino acids 1192-1457) and the core or nucleocapsid structural protein (HCV-1 amino acids 1-150). HC43 is expressed in E. coli as a fusion protein by using a plasmid (pKRR826) containing the pL promoter of bacteriophage lambda (described in U.S. Pat. No. 6,846,905), utilizing a codon-optimized sequence from the HCV H strain (i.e., HCV-1; Ogata et al., PNAS USA 88: 3392-3396 (1991)). Two non-HCV coding amino acids separate the NS3 and core sequences. There are commercially marketed anti-HCV assays using such a fusion protein. The expression of this fusion protein in E. coli via a temperature inducible system results in the formation of insoluble inclusion bodies. These must be solubilized with urea, reductant and SDS in order to obtain pure, monomer protein for use in the immunoassay (as solid phase antibody capture reagent). Derivatives of this protein (e.g. 9MB31) disclosed in US patents owned by Abbott comprise truncated core protein sequences and are expressed in temperature inducible systems yielding protein that is insoluble.
Another such recombinant protein used for the detection of anti-HCV antibodies is C100. This recombinant protein is derived from the NS3 and NS4 regions of the HCV genome (HCV amino acids 1569-1931), and is expressed in yeast with an N-terminal superoxide dismutase (SOD) fusion of 527 amino acids (see, e.g., U.S. Pat. No. 5,350,671). Although 363 amino acids of the HCV genome are present in the recombinant protein, studies have demonstrated that the majority of antibody binding occurs in two smaller regions within the NS4 region. The first region is the 5-1-1 region, which comprises HCV amino acids 1691-1733, and the second is the C100 region made up of HCV amino acids 1921-1940.
Other NS3 helicase constructs used for immunoassay development have been described by Jin and Petersen (Archives or Bioch Biophys, 1995, 323:47-53; Sallberg et al., 1996, J Gen Virol, 77:2721-2728; Chien et al. 1998, Hepatology, 28:219-224) but these constructs encompass residues 1207-1612 and do not included the full length helicase (1207-1657). In addition, the aforementioned proteins are again expressed in insoluble form and purified under denaturing conditions and require protein refolding techniques in order to regain enzymatic activity, prior to their use in immunoassays.
Many HCV diagnostic assays make use of an NS3 antigen, in different forms. HCV NS3 is a multifunctional protein, containing a serine protease domain within its N-terminal third and an NTPase/helicase domain within its C-terminal two-thirds. Polynucleotide-stimulated NTPase activity, capable of hydrolyzing all NTPs and dNTPs, has been shown, while RNA helicase activity, requiring ATP and a divalent ion, has also been identified: the NS3 C-terminal domain is capable of unwinding RNA-RNA, RNA-DNA and DNA-DNA substrates in a 3′-5′ direction.
Crystal structure analysis of the HCV NS3 helicase has shown that this enzyme is composed of three domains. Domain I (approximately residues 181-326 of NS3) and Domain II (approximately residues 327-481 of NS3) have little sequence identity, but share similarities in structure being composed of a large central β-sheet flanked by α-helices, and are homologous in structure to the central region of the RecA protein. Domain III (approximately residues 482-631 of NS3) is mostly α-helical and contains part of the single-stranded nucleic acid binding site. Domains I and III share a more extensive interface than either share with Domain II. Therefore, Domains I and III form a rigid unit, whereas Domain II is connected to Domains I and III by solvent-exposed polypeptide segments capable of supporting large scale, relative rotations of Domain II. In particular, an unusual molecular feature is a long antiparallel β-loop that extends from the central β-sheet of Domain II to Domain III where the end of the loop becomes an integral part of the domain III structure. Thus, similar to other helicases, domain motions are characteristic for the activity of the HCV helicase (see Gu & Rice, PNAS, 2010, 107:521-528 and references therein).
While there are some commercially available assays for serological determination of HCV infection using NS3 antigens these assays still need improvement to allow their use for detection earlier within the HCV infection window. Thus, there remains a need for additional assays having increased sensitivity by reducing the HCV antibody seroconversion window. The present invention addresses this need by providing improved sensitivity of anti-NS3 detection in such serological assays.